Efficacy and immunological changes of sublingual immunotherapy in pediatric allergic rhinitis

Background Allergen-specific immunotherapy, including subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT), improves the disease progression of allergic rhinitis (AR). SCIT and SLIT exhibit similar efficacy, but SLIT has less systemic reactions. However, few studies have investigated the underlying mechanisms of SLIT treatment. In this study, we explored the efficacy of SLIT under different treatment durations and immunological changes. Methods This retrospective study was conducted from August 2017 to August 2022 in our hospital. A total of 314 children who underwent SLIT were divided into the following groups based on their treatment duration: the 1 year group (6 months–1 year), the 2 years group (1–2 years), and the 3 years group (2–3 years). The treatment efficacy was confirmed using a combined symptom and medication score (SMS). Multiple serum cytokines were measured using Luminex. Various immune cells in PBMCs were determined using flow cytometry. Results The total nasal symptom score (TNSS), rescue medication score (RMS), and SMS of the 3 years group was significantly different from those of the 1 years and 2 years groups. At the end of the 2 years following cessation of SLIT, the following results were observed in the 3 years group: 1) the TNSS, RMS, and SMS had significantly improved, 2) the serum IL-10, TGF-beta, and IL-35 levels had increased significantly, and 3) the percentages of regulatory T cell, regulatory B cell, and follicular regulatory T cell increased significantly. Conclusion Our results suggest that 3 years of SLIT is necessary for long-term effects and continued immunological changes.


INTRODUCTION
The incidence of allergic rhinitis (AR) has significantly increased in China over the last few decades. 1,2 A recent study reported that the occurrence of AR ranged from 9.6% to 23.9% in 18 major cities in China. 1 Allergen avoidance, medications aimed to relieve symptoms, allergen-specific immunotherapy (AIT), and health education are 4 methods of comprehensive treatment. 3 AIT, including subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT), is currently the only treatment to improve the disease progression of AR. 4 Compared with SCIT, SLIT has similar efficacy and less systemic reactions. 5,6 Several studies have proven that 3 years of AIT induces long-lasting effects, even after cessation. [7][8][9] The mechanisms of successful AIT include reduced allergen specific T-and B-cell inflammation, decreased IgE production, and inhibited mast cell and basophil activation, while the IgG4 production, the function of T regulatory cells and B regulatory cells were enhanced. 10 However, few studies have investigated the underlying mechanisms of treatment with SLIT in children.
In this study, we aimed to explore the efficacy of SLIT under different treatment durations and immunological changes.

METHODS Patients
We conducted a retrospective study from August 2017 to August 2022 in our hospital. The AR was determined according to the Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines. 11 This included typical allergic symptoms such as runny nose, itchy nose, sneezing, nasal block, positive skin-prick test (SPT), and/or serumspecific immunoglobulin E (IgE) for Dermatophagoïdes farina or accompanied by Dermatophagoïdes pteronyssinus. All recruited subjects were children (4-18 years old) and the disease duration was longer than 1 year. Children with asthma, nasal polyposis, immunodeficiency, severe systematic diseases, or sensitivity to allergens except for Dermatophagoïdes farina or Dermatophagoïdes pteronyssinus were excluded. Asthma was excluded using questionnaires on the history of recurrent dyspnea, wheezing, or cough episodes, and methacholine PC20 4 mg/mL or albuterol reversibility of FEV1 ! 10%. Informed consent was obtained from the children and their parents. Our study obtained approval from local ethics committee boards.

Immunotherapy and grouping
For SLIT, Dermatophagoides farinae drops (Zhejiang Wolwo, China) were provided. The drugs were administered with increasing concentrations from 1 mg/mL to 333 mg/mL over the course of 3 weeks according to the recommendations (1,2, 3, 4, 6, 8, 10 drops of No. 1 to No. 3 drops every week for 3 weeks). In the maintenance stage from the fourth week, 333 mg/mL of drugs (3 drops) were given until the end of the treatment. All of the children's guardians were provided with a detailed and comprehensive health education and were informed the importance of enough treatment period (3 years) and regular follow-up. After 3 years' follow-up, the children were divided into 3 groups according to actual completed treatment duration: 1 year group (6 months-1 year), 2 years group (1-2 years), and 3 years group (2-3 years).

Efficacy of SLIT
The treatment efficacy and the adverse events of SLIT were assessed using symptom and medication scores at the baseline, at the end of SLIT and at the end of the 2 years following cessation of SLIT using Diary Card throughout the study (at least 3 times every week). 12 In brief, the rhinorrhea, sneezing, nasal obstruction, and itchy nose were scored from 0 to 3 according to disease severity (0, no symptom; 1, slight symptoms; 2, moderate symptoms; 3, severe symptoms) and the total nasal symptom score (TNSS) was obtained. The rescue medication score (RMS) was defined as 1 point for use of oral or topical antihistamines and 2 points for topical corticosteroids. The combined symptom and medication score (SMS) was used for evaluation of efficacy. The children obtained a 30% SMS decrease compared to their baseline score were defined as response group.

Collection of serum samples and measurement of Ig and cytokines in serum
Venous blood samples were collected after centrifugation at 3000 g for 20 min. The serum was stored at À80 C for further analysis. Multiple serum cytokines were tested by Luminex. The Ig levels were measured by enzyme linked immunosorbent assay (ELISA) kits (R&D systems, USA) according to the instructions.

Flow cytometry analyses
Isolation of the peripheral blood mononuclear cells (PBMCs) was performed by density-gradient centrifugation. Various immune cells in PBMCs were determined using flow cytometry. The antibodies used for staining are listed in Table 1.

PBMC stimulation
The isolated PBMCs (2*10 6 cells) cultured in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific) containing 10% fetal bovine serum, 100 U/ml of penicillin, and 100 mg/ mL of streptomycin (Thermo Fisher Scientific) were incubated with 10 mg/mL recombinant Der p 1 (Indoor Biotechnologies Ltd) at 37 C for 5 days. The supernatants were collected for cytokine measurement by Luminex. The cell viability was 98%-100% as determined by Trypan blue exclusion assay. After washing with 2% FBS-containing PBS, cells were stained with fluorescence-labeled Abs (Table 1) for 30 min, washed with PBS, and analyzed by the FACSAria II flow cytometry device (BD Biosciences).

Statistical analysis
Statistical analysis was performed using Graph-Pad Prism 9.0 software. A preliminary analysis of the data was performed using normality, homogeneity of variances, firstly. Comparisons between groups were performed by unpaired, nonparametric testing (Kruskal-Wallis test) and chisquare tests. The Intra-Group comparison was performed using a paired t-test. P < 0.05 was defined as statistically significant.

Baseline characteristics
A total of 314 children were enrolled in this study and 296 children finished the study. The baseline characteristics of subjects are summarized in Table 2. The age, sex ratio, BMI, and disease duration and severity among different groups had no significant difference (P > 0.05).
For the 1 year group of 107 children, 101 of them responded to our survey at 2 years after the cessation of treatment. For the 2 years group of 74 children, 70 of them responded to our survey at 2 years after the cessation of treatment. Lastly, for the 3 years group of 134 children, 125 of them responded to our survey at 2 years after the cessation of treatment. No child developed asthma during the study period. The reasons for not being able to persist in treatment in 1 year and 2 years group were summarized in Table S1.

Comparison of efficacy among groups
The TNSS, RMS, and SMS at the end of SLIT improved significantly compared with the baseline levels in all groups (Table 3). At the end of SLIT, the TNSS, RMS, and SMS of the 1 year group was significantly higher than the 2 years and 3 years groups, but there was no difference between the 2 years and 3 years groups (Table 3). At the end of the 2 years following cessation of SLIT, the TNSS, RMS, and SMS of the 3 years group had significant improved compared to the 1 year and 2 years groups. The adverse events were

Cells
Antibodies Company   Table 3 and no severe adverse events were reported.

Comparison of Ig and cytokines among groups
The serum IL-10, TGF-beta, and IL-35 levels at the end of SLIT and the end of the 2 years following cessation of SLIT in the 3 years group increased significantly compared to the 1 year and 2 years groups (Fig. 1). At the end of the 2 years following cessation of SLIT, the serum IL-10, TGFbeta, and IL-35 levels in the 3 years group were not significantly changed (Fig. 1). The sIgE, IgG, IL-4, IL-5, IL-13, IL-17, and IL-21 levels were not significantly different between groups (Fig. 1). Despite the IL-4 expression decreased at the end of 2 years following cessation, no significant difference was found.

Comparison of percentage of immune cells among groups
The percentages of regulatory T cell (Treg), regulatory B cell (Breg), and follicular regulatory T cell (Tfr) at the end of SLIT and the end of the 2 years following cessation of SLIT in the 3 years group increased significantly compared to the 1 year and 2 years groups (Figs. 2 and 3).  (Fig. 2). In the 3-year group, ILC2 showed a downward trend at the end of treatment and then moderated but remained lower 2 years after cessation, but no significant difference was found.

Comparison of cytokines and percentage of immune cells between good responsive and poor responsive groups
To analyze the coorelation between long-term efficacy of SLIT and immunological changes, we compared the immunological parameters between the good responsive and poor responsive groups at the end of the 2 years following cessation of SLIT. Our data showed that the serum IL-10, TGF-beta, IL-35 levels and the percentages of Treg, Breg, Tfr were significantly lower in poor responsive groups compared with good responsive group (Table 4), while other parameters showed no significant difference between groups.

Allergen-induced immunological responses
After stimulated by Recombinant Der p 1, the serum IL-10, TGF-beta, IL-35 levels and the percentages of Treg, Breg, Tfr in the 3 years group were significantly higher compared with baseline levels (Table 5), while other cytokine levels and the percentages of other cells showed no significant difference between groups (Data not shown).

DISCUSSION
Stable clinical benefits from SLIT often require at least 3 years of treatment. For example, Xu's study reported that 3 years of SLIT was more efficacious than 1 year or 2 years of SLIT in AR patients allergic to house dust mites (HDM). 13 Moreover, another study suggested that 4 years of SLIT is the optimal choice for long-lasting effects in HDMinduced respiratory disease. 14 Consistently, our data found that children obtained more beneficial results in the alleviation of clinical symptoms and medication usage at the end of 2 or 3 years of SLIT than 1 year of SLIT. However, there were no significant differences between the 2 years and 3 years groups at the end of treatment in the improvement of clinical symptoms and medication usage. This suggests that 2 and 3 years of SLIT may produce similar clinical benefits during the treatment. Next, we compared the TNSS, RMS, and SMS score 2 years at the end of the 2 years following cessation of SLIT and found that only children in the 3 years group obtained long-lasting effect, while more than 50% of the children in the 1 year and 2 years groups rebound after the cessation of treatment. These results confirm the significance of long-term SLIT in maintaining long-lasting effects.
Immune cells and inflammatory cytokines composed of complex networks are involved in the pathogenesis of AR and SLIT. Th2 cells produce IL-4 and IL-13, which contribute to the differentiation of Th2 cells and IgE production. 15,16 In the late-phase, Th2 cytokines and tissue eosinophilia exacerbate allergic inflammation. 16 In SLIT, IL-5 þ , IL-13 þ , and CD4 þ cells may be used as biomarkers for successful SLIT as described in a previous study. 17 Group 2 innate lymphoid cells (ILC2s), another main source of IL-5 and IL-13, also stimulated allergic responses and play a key role in the development of AR. Interestingly, only IL-10 þ and ILC2s were found to influence the mechanism of allergen immunotherapy. 18 Tfh cells also have the ability to assist B cells to produce IgE. 19 The percentage of peripheral Tfh2 cells was positively related to IgE levels in AR. 19 After AIT, decreased Der p 1-specific IL-4 Tfh cells was correlated with the alleviation of clinical symptoms. 19 The change of specific IgE and IgG4 levels during SLIT has also received much attention, especially the serum specific IgE/total IgE ratio, which was believed to be a good biomarker for the clinical efficacy of AIT. 20 Interestingly, our data found that the percentages of Th2, Th17, Tfh2, ILC2, and related cytokines as well as Ig levels were not changed significantly during SLIT. These discrepancies between children and adults may be explained by several possible reasons: immaturity of immune system in children leads to different immune tolerance mechanisms, different major allergen type in different study population (eg, mite vs cedar pollinosis), or the mode of AIT (SCIT vs SLIT).
In the 1 year and 2 years group, we found no change of the anti-inflammatory cytokine levels and the percentage of suppressive cells, suggesting that 1 or 2 years of SLIT may be not sufficient to induce immune tolerance. Interestingly, some previous reported immunal changes after 1 year of AIT. [21][22][23][24][25] However, the different allergen type (mite vs cedar pollinosis), treatment method (SCIT vs SLIT), and treatment population (adult vs children) may explain the different results. Our data also showed that the percentages of Treg, Breg, and Tfr in the 3 years group increased significantly. Consistently, Terada et al found that the percentages of Foxp3 þ , Treg cells, and Breg cells in PBMCs were significantly elevated after 4 years of SLIT treatment for cedar pollinosis. 23 Moreover, IL-35 induced Treg cell counts were increased in patients receiving SLIT compared to patients with AR. 26 Tfr, a new effector subset of Treg, can limit B cell activation and IgE production. 27 After AIT, the numbers and function of Tfr were enhanced, which is consistent with our results. 28 Consistently, we also found that anti-inflammatory cytokines and cells were up-regulated in good responsive group compared with poor responsive groups at the end of the 2 years following cessation of SLIT. In the allergen-induced immunological responses, we also found that SLIT improved the ability of antiinflammatory cytokines secretion and the proliferation of suppressive cells. After 3 years of SLIT, the Der p 1 induced serum IL-10, TGF-beta, IL-35 expression, and peripheral blood of Treg, Breg, Tfr ratio were increased significantly. These antiinflammatory cytokines and suppressive cells contribute to the inhibition of allergic inflammation and corresponding symptoms.
Collectively, our data suggest that effective SLIT may be attributed to the induction of suppressive cells rather than the inhibition of inflammatory cells. Moreover, these immunological changes were not altered even two years after the cessation of the treatment, suggesting that 3 years of SLIT can establish long-term stable immune tolerance.

CONCLUSION
In summary, our results suggest that at least 3 years of SLIT treatment is necessary for long lasting effects and continued immunological improvements. During SLIT, suppressive cells such as Treg, Breg, and Tfr may play more important roles than inflammatory cells. Further studies are required to explore these results.